Staphylococcus aureus is a prominent pathogen in today's society. It has been implicated in a number of potentially lethal diseases. In the past two decades, the worldwide spread of methicillin-resistant S. aureus strains has caused a significant clinical problem. The infections due to MRSA can be treated only with the glycopeptide, vancomycin. The recent emergence of almost untreatable vancomycin-resistant S. aureus poses a major and immediate threat to public health. Thus, there is an urgent need for the development of new antimicrobial agents to treat staphylococcal infections. In our lab we have been using various approaches such as isolation and characterization of laboratory-derived GISA strains, along with proteomic and genomic analyses to investigate the mechanisms of vancomycin/ methicillin resistance in S. aureus. Transcriptional profiling of S. aureus cells treated with vancomycin showed that about 160 genes were over-expressed and 150 genes were under-expressed. Among them the vraSR, a two-component system, has been reported by several investigators to play a major role in controlling the expression of many cell wall stress stimulon genes. However, the precise mechanism by which vraSR senses the antibiotic stress and transduces the signal to the target genes is still not clear. The main objective of this proposal is to further analysis of the VraSR system of S. aureus. The objective will be accomplished by 1) Cloning and overexpression of vraR and characterizing biochemical properties of VraR with respect to DNA binding specificity using EMSA and DNase protection assays. The purified VraR protein will also be used for crystal structural studies to determine the role of key residues in VraR in DNA protein interactions (Diffraction data will be collected in collaboration with Dr. Deivanayagam, Center for Macromolecular Crystallography Laboratory, UAB, AL. 2) Identification of the genes which are under the regulation of VraSR using chromatin immunoprecipitation assay and construction of mutation in some of those genes to determine their effect on antibiotic sensitivity. Completion of this proposal will provide us with new insight into the role and identity of VraSR regulated genes in S. aureus. We believe the knowledge of these genes and their functions may be critical for developing new strategies for controlling these bacteria, and some of the genes identified may provide novel targets for antimicrobial agents. PUBLIC HEALTH RELEVANCE: S. aureus is a prominent pathogen and a cause of significant clinical problem due to the development of antibiotic resistance including vancomycin. In this study we propose to further analyze the vraSR, the two component system which is involved in development of antibiotic resistance. We believe the knowledge of this study may aid in developing new strategies for controlling these bacteria and some of the genes identified may provide novel targets for antimicrobial agents. [unreadable] [unreadable] [unreadable]